inhibition of autophagy ameliorates pulmonary microvascular dilation and pmvecs excessive proliferation in rat experimental hepatopulmonary syndrome

In the present study, we report that autophagy was activated in common bile duct ligation CBDL rats and cultured pulmonary PMVECs induced by CBDL rat serum, two accepted in vivo and in

Inhibition of autophagy ameliorates pulmonary microvascular dilation and PMVECs excessive proliferation

in rat experimental hepatopulmonary syndrome

Duo Xu1, Bing Chen1, Jianteng Gu1, Lin Chen1, Karine Belguise2, Xiaobo Wang2, Bin Yi1 & Kaizhi Lu1

Hepatopulmonary syndrome (HPS) is a defective liver-induced pulmonary vascular disorder with massive pulmonary microvascular dilation and excessive proliferation of pulmonary microvascular endothelial cells (PMVECs) Growing evidence suggests that autophagy is involved in pulmonary diseases, protectively or detrimentally Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS In the present study, we report that autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary PMVECs induced by CBDL

rat serum, two accepted in vivo and in vitro experimental models of HPS Furthermore, pharmacological

inhibition of autophagy with 3-methyladenine (3-MA) significantly alleviated pathological alterations

and typical symptom of HPS in CBDL rats in vivo, and consistently 3-MA significantly attenuated the CBDL rat serum-induced excessive proliferation of PMVECs in vitro All these changes mediated by 3-MA

might explain the observed prominent improvement of pulmonary appearance, edema, microvascular

dilatation and arterial oxygenation in vivo Collectively, these results suggest that autophagy activation

may play a critical role in the pathogenesis of HPS, and autophagy inhibition may have a therapeutic potential for this disease.

Hepatopulmonary syndrome (HPS) is a life-threatening disease characterized by a triad of chronic liver disease (CLD), intrapulmonary vascular dilation (IPVD) and serious hypoxemia1,2 The prevalence of HPS varies from 4–47% due to different cut-offs in defining arterial hypoxemia in primary studies, and its mortality rate is about 41%3,4 Although progress has been made in delineating the mechanisms underlying the imbalance of vasoactive substances, pulmonary vascular alterations and angiogenesis in HPS, to date, there is still lack of effective thera-peutic approaches apart from liver transplantation (LT)5–7

Autophagy (derived from the Greek words meaning “self eating”) is identified as an evolutionarily conserved cellular housekeeping process that is involved in the degradation of protein and organelle8,9 Increasing evidence has demonstrated that the dysfunction of autophagy contributes to various diseases, such as cancer, atheroscle-rosis, Alzheimer’s disease and acute lung injury (ALI)10–13 In certain circumstances, autophagy plays a protective role via its clearing the damaged and unhealthy organelles For example, autophagy is involved in stress adaption

in lung injury through its removal of the damaged organelles and thus the promotion of cell survival14 However, the massive and persistent activation of autophagy may contribute to excessive cell proliferation and pathologi-cal angiogenesis15,16 HPS is a kind of pulmonary vascular complication with high mortality rate17 Considering that autophagy is involved in and critical in lung injury, protectively or detrimentally Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS However, it is still unclear how the process of autophagy is altered in the pathogenesis of HPS and how this alteration is detrimental or beneficial

to HPS

1Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

2University P Sabatier Toulouse 3 and CNRS, LBCMCP, 31062 Toulouse Cedex 9, France Correspondence and requests for materials should be addressed to B.Y (email: yibin1974@163.com) or K.L (email: lukaizhi2013@163.com)

Received: 07 April 2016

accepted: 11 July 2016

Published: 02 August 2016

OPEN

At the cellular level, our previous research demonstrated that the common bile duct ligation (CBDL) rat serum

induces the excessive proliferation of pulmonary microvascular endothelial cells (PMVECs) in vitro, which could

contribute to the HPS-associated angiogenesis, a highly complicated and regulated process to form new ves-sels and capillary networks18–20 In the present study, we hypothesized that the initial autophagy activation may play a pivotal role in the pathological alterations of HPS To address this hypothesis, we assessed whether auto-phagy level is increased in both CBDL rats and cultured PMVECs under the stimulation of CBDL rat serum,

and whether autophagy inhibition could affect the pathological status of HPS in both in vivo and in vitro models.

Results Activation of autophagy in lung tissues of CBDL rats Autophagy status is defined by the presence and number of autophagosomes within cells Thus, we assessed autophagy status by the detection of autophago-somes in lung tissues of CBDL rats with transmission electron microscopy (TEM) Autophagoautophago-somes, which are the double membrane structures or double membrane vacuoles, were prominently observed in lung tissues of 2- and 4-week CBDL rats (Fig. 1A, as indicated by broad arrows), while they were weakly detectable in the con-trol sham rats The most abundant autophagosomes were observed in 2-week CBDL rats, and there is a slightly decrease in 4-week CBDL rats Next, we determined the expression levels of autophagy-related proteins (LC3B, Beclin-1 and P62) in lung tissues of CBDL rats, as evaluated with Western blotting LC3B is required for the for-mation of autophgosome membranes, and upon induction of autophagy cytosolic LC3-I is cleaved and lipidated

to form LC3-II Beclin-1 forms a protein complex with the Class III PI3K, which is critical for the recruitment of LC3B p62 degradation reflects the lysozyme fusion and autophagosome breakdown So autophagy activation is commonly evidenced by the increase in LC3-II/LC3-I and Beclin-1 protein levels, and reduction in p62 protein levels8 Our results demonstrated that the protein levels of LC3B and Beclin-1 in the lung tissues were significantly increased and peaked at 2-week CBDL rats; oppositely, the protein levels of p62 in lung tissues were significantly decreased and dropped to minimal in 2-week CBDL rats; the positive or negative changes of all these proteins showed a slightly reduction in 4-week CBDL rats, and all these temporal changes are consistent with our observa-tion of autophagosome formaobserva-tion (Fig. 1B) Both the formaobserva-tion of autophagosomes and the respective changes of autophagy-related proteins demonstrated the activation of autophagy in lung tissues of CBDL rats

Activation of autophagy in cultured PMVECs under the stimulation of CBDL rat serum Because PMVECs is a major cell type in pulmonary microvasculature and our previous research has demonstrated that the excessive proliferation of PMVECs contributes to the development of HPS20,21 Thus, in this part we asked

whether autophagy activation observed in vivo could be also detected in the in vitro cultured cell model The

formation of autophagosomes in cultured PMVECs, under the stimulation of normal rat serum or CBDL rat serum for 0 h (T1), 12 h (T2) and 24 h (T3), was detected with TEM The control PMVECs contained normal organelles, nucleus and chromatin After the stimulation with CBDL serum for different time points, PMVECs were observed to contain a number of typical autophagosomes in the cytoplasm (Fig. 2A, as indicated by broad arrows) The most abundant autophagosomes were observed at 24 h after the stimulation of CBDL rat serum In addition to the typical autophagosomes with engulfed organelles, the fusion of autophagosomes with lysosomes was occasionally detected in PMVECs after the CBDL rat serum stimulation (Fig. 2A, as indicated by asterisk) The expression levels of autophagy-related proteins (LC3B, Beclin-1 and P62) in cultured PMVECs, under the stimulation of normal rat serum or CBDL rat serum, were evaluated with Western blotting and immunocyto-chemistry Our results demonstrated that the protein levels of LC3B and Beclin-1 in cultured PMVECs were significantly increased and peaked at 24 h after the CBDL rat serum stimulation, while the protein levels of p62 were significantly decreased and dropped to minimal at 24 h after the CBDL rat serum stimulation (Fig. 2B,C) Since autophagy is a dynamic cellular process, we analyzed the autophagic flux status of PMVECs under the stimulation of normal rat serum or CBDL rat serum for 24 h, as evidenced with the spatial distribu-tion of mCherry red fluorescent protein (mRFP) and green fluorescent protein (GFP) from the expression of mRFP-GFP-LC3 adenovirus (Fig. 2D) Based on the pH sensitivity difference between GFP and mRFP, neutral autophagosomes and acidic autolysosomes are labeled with yellow color or red color in merged images respec-tively22,23 The numbers of GFP and mRFP dots in PMVECs were significantly increased after CBDL rat serum stimulation In the merged images, both numbers of yellow dots and free red dots were increased, indicating that the accumulation of both autophagosomes (yellow puncta) and autolysosomes (red puncta) was induced in PMVECs after CBDL rat serum stimulation These findings further demonstrated that CBDL rat serum could induce autophagy activation in cultured PMVECs Taken together, we can conclude that autophagy is activated

in both in vivo and in vitro models of HPS.

Inhibition of autophagy improved pulmonary appearance, histology, microvascular dilata-tion and arterial oxygenadilata-tion in CBDL rats The main question is whether activation of autophagy might play an important role in the pathology of HPS Thus, we evaluated whether autophagy inhibition influ-enced the pathological alterations and typical symptom of HPS Firstly, we confirmed the inhibitory effect of 3-methyladenien (3MA), the widely used inhibitor of autophagy 3MA(15 mg/kg) effectively blocked autophagy activation in CBDL rats, as evidenced by the decreased protein levels of LC3B and Beclin-1 as well as the increased protein levels of p62 (Fig. 3A) The pathological alterations of HPS are evaluated by pulmonary appearance, his-tology and microvascular dilatation24 Therefore, we next assessed the effect of autophagy inhibition on all these

3 standards with typical lung tissue samples, pulmonary wet-to-dry ratio, haematoxylin and eosin (HE) staining, fluorescent-labeled microsphere assay and TEM Compared with the sham group, 2- and 4-week CBDL rats had serious pulmonary hemorrhage, old petechial, edema and microvasular injuries, while 3MA administration sig-nificantly alleviated these histological alterations (Fig. 3B–F) Similarly, arterial blood gas analysis demonstrated

that both 2- and 4-week CBDL rats had the decreased levels of PaO 2 and the increased levels of A a PO, while 3MA

treatment strongly improved pulmonary oxygenation function (Table 1)

Inhibition of autophagy attenuated the excessive proliferation of PMVECs induced by CBDL rat serum Since the pathological process of HPS is highly related with the excessive proliferation of PMVECs,

we asked whether the inhibition of autophagy activation might alleviate the induced proliferation of PMVECs

To determine the role of autophagy in CBDL rat serum-induced excessive proliferation of PMVECs, cultured PMVECs were treated with autophagy inhibitor 3-MA 3MA(5 mM) treatment effectively blocked autophagy activation in cultured PMVECs at 24 h after CBDL rat serum stimulation, as evidenced by the inhibition of auto-phagosome formation, the reduced protein levels of LC3B and Beclin-1 as well as the increased protein levels of P62, and the repression of autophagic flux with the much lower amounts of autophgic structures (Fig. 4A–C) Next, CCK-8 analysis was applied in order to check the effect of autophagy inhibition on cell proliferation abil-ity CCK-8 analysis demonstrated that autophagy inhibition with 3MA treatment efficiently repressed CBDL rat serum-induced proliferation of PMVECs at each time-point (Fig. 4D) These findings suggest that autophagy activation may contribute to CBDL rat serum-induced excessive proliferation of PMVECs, and 3MA can attenu-ate this alteration through inhibiting the autophagy activation

Figure 1 Activation of autophagy in lung tissues after common bile duct ligation (CBDL) in vivo

(A) Representative transmission electron microscopy (TEM) images and graphical summaries of autophagosomes

in lung tissues of sham, 2- and 4-week CBDL rats (n = 5) Broad arrows represent autophagosomes 10 fields for each

rat were observed (B) Western blotting and graphical summaries of LC3B, Beclin-1 and p62 protein levels in lung

tissues of sham, 2- and 4-week CBDL rats (n = 5) All blots were representative of three independent experiments Values were expressed as means ± SEM * P < 0.05 compared with sham.

HPS, a defective liver-induced pulmonary vascular disorder, is characterized by worsening hypoxemia due to intrapulmonary vascular dilatation (IPVD), arteriovenous malformations and increased vasoactive substances

in the setting of chronic liver disease (CLD)17,25 Over the past two decades, the pathogenesis and precise mecha-nisms of HPS were under active investigation Although much progress has been made in delineating the mech-anisms underlying the imbalance of vasoactive substances, pulmonary vascular alterations and angiogenesis in HPS, additional mechanisms may involve in this disease26–29 Recently, many studies have demonstrated that autophagy is involved in various diseases, especially lung diseases30–33 So it is interesting and important to check whether autophagy might be related to HPS and whether autophagy plays a critical role in HPS Our present work

is the first one to evaluate autophagy activation in the development of HPS

Figure 2 Activation of autophagy in cultured PMVECs under the stimulation of CBDL rat serum in vitro

(A) Representative TEM images and graphical summaries of autophagosomes in cultured PMVECs under

the stimulation of normal rat serum or CBDL rat serum for 0 h (T1), 12 h (T2) and 24 h (T3) Broad arrows represent autophgosomes Asterisks represent autolysosomes 30 random cells for each group were observed

(B) Western blotting and graphical summaries of LC3B, Beclin-1 and p62 protein levels in cultured PMVECs

under the stimulation of normal rat serum or CBDL rat serum for 0 h (T1), 12 h (T2) and 24 h (T3) All blots

were representative of three independent experiments (C) Representative immunocytochemistry images of

LC3B, Beclin-1 and p62 protein in cultured PMVECs under the stimulation of normal rat serum or CBDL rat

serum for 24 h (D) Representative confocal microscope images and graphical summaries of LC3 in different

groups of PMVECs infected with mRFP-GFP-LC3 adenovirus for 24 h 30 random cells for each group were observed Values were expressed as means ± SEM * P < 0.05.

Figure 3 Inhibition of autophagy with 3-methyladenien (3MA)-15 mg/kg improved pulmonary appearance,

histology and microvascular dilatation after CBDL in vivo (A) Western blotting and graphical summaries of

LC3B, Beclin-1 and p62 protein levels in lung tissues of sham, 2- and 4-week CBDL rats with or without 3MA

administration (n = 5) All blots were representative of three independent experiments (B) Representative

pictures of lung tissue samples in sham, 2- and 4-week CBDL rats with or without 3MA administration 3MA

alleviated pulmonary hemorrhage and old petechial in 2- and 4-week CBDL rats (C) Graphical summaries of

pulmonary wet-to-dry ratio in sham, 2- and 4-week CBDL rats with or without 3MA administration (n = 5) 3MA improved pulmonary edema in 2-week CBDL rats, as evidenced by the decreased pulmonary wet-to-dry ratio The ratio in the 4-week CBDL rats decreased to the baseline, and there is no significant difference between

the 4-week CBDL rats with or without 3MA administration (D) Representative micrographs of haematoxylin

and eosin staining of pulmonary microvessels (indicated by arrows) in sham, 2- and 4-week CBDL rats with

or without 3MA administration 3MA alleviated the disorganized and enlarged microvessels in 2- and 4-week

CBDL rats (E) Graphical summaries of fluorescent-labeled microsphere assay in sham, 2- and 4-week CBDL rats

with or without 3MA administration (n = 5) 3MA improved pulmonary microvasular dilation in 2- and 4-week

CBDL rats, as evidenced by the decreased ratio of brain-over-lung (F) Representative TEM images of lung tissues

in sham, 2- and 4-week CBDL rats with or without 3MA administration 2- and 4-week CBDL rats had enlarged microvessels filled with erythrocytes and destructive pulmonary microvasculature with exfoliated alveolar epithelium, respectively (indicated by arrows) 3MA significantly alleviated the above alterations Values were expressed as means ± SEM * P < 0.05 compared with sham #P < 0.05 compared with CBDL 2-week ##P < 0.05

compared with CBDL 4-week

Autophagy, a self-digestion and dynamic process, is involved in long-lived proteins and dysfunctional orga-nelles degradation34,35 The basal level of autophagy is essential for homeostasis, and the altered autophagy has been demonstrated in various pathological alterations Although the beneficial effects of autophagy after lung injury have been demonstrated by some studies, in some cases, pharmacological inhibition of autophagy after some insults has protective effects16,36–38

Our in vivo data suggest that autophagy was over-activated at the early time point (2-week) after CBDL and

has a slight recovery at the later time point (4-week) This is a time-dependent alteration in autophagy levels after CBDL, which indicates that autophagy may have a certain-limited time course in the development of HPS

We expect that the over-activated autophagy in the early time after CBDL contributes to the development of HPS And, in the present study we found that 3-MA treatment from the 1st day and end on the 2nd weeks post

CBDL significantly blocked autophagy activation and improved the pathological alterations in vivo, which further

demonstrate this point Therefore, we propose that an early intervention aimed at the reduction in autophago-somes accumulation has the protective effects after CBDL However, it is worth noting that a prolonged treatment with 3-MA has been demonstrated to promote autophagy flux under nutrient-rich conditions due to its differ-ential temporal effects on class I and class III PI3K39 Such mechanism gives us an insightful thinking that the effect of 2-week 3-MA administration may be different from that of 4-week In addition, it is still unclear why the autophagy status has a slight recovery in the late stage of CBDL rats and whether this alteration plays a regulatory role in HPS All these important and interesting sections mentioned above need further research

Our in vitro data further demonstrate that the above protective effect may result from the inhibition of PMVECs excessive proliferation and pulmonary microvascular dilation Takeshi et al demonstrated that

auto-phagy gets induced in ECs in response to the pro-apoptotic agent, sulforaphane, and the inhibition of autoauto-phagy potentiates the pro-apoptotic effect40 Their findings open premises for the use of autophagy inhibitors in com-bination with anti-angiogenic agents, and also enlighten us about that autophagy may play a regulatory role in PMVECs excessive proliferation during HPS progression In the present study, we demonstrated, for the first time, that autophagy activation may contribute to CBDL rat serum-induced proliferation of PMVECs, and 3-MA attenuates this alteration through inhibiting the autophagy activation This finding provides another important therapeutic strategy for HPS The development of autophagy inhibitors with higher specificity for ECs as well as angiogenic endothelium is desired to be tested in clinical trials

Based on the above findings, we have a deep thinking about why autophagy was activated after CBDL and how autophagy modulates PMVECs proliferation Here, we give some speculation about these questions In recent years, autophagy regulation is under active investigation It is worth noting that some common stimuli for auto-phagy activation, such as hypoxia, ER stress and inflammatory mediators, are also involved in the pathogenesis of HPS8,41,42 Moreover, Li et al demonstrated that in bacteria-induced lung injury, Annexin A2 induces autophagy

activation through inhibiting Akt1-mTOR-ULK1/2 signaling pathway43 Coincidently, our previous research found that CBDL rat serum induces Annexin A2 expression, which further contributes to the HPS-associated angiogenesis through ERK1/2 and NF-kB signaling pathway44 So the above factors might be responsible for the autophagy activation after CBDL About the modulation of PMVECs proliferation, the interplay of autophagy and apoptosis should be considered Autophagy (‘self-eating’) and apoptosis (‘self-killing’) determine the turn-over of cytoplasmic organelles and entire cells, respectively Although both autophagy and apoptosis are under the control of multiple common upstream signals, these processes also cross-regulate each other, mostly in an inhibitory manner As such mechanism, autophagy activation can reduce cell death through selectively inhibiting the abundance of pro-apoptotic proteins, which further promotes cell proliferation45 This is an interesting part and we need more support data to demonstrate it in the following research

Another interesting question is whether the effect of autophagy inhibition comes from inhibition of

intrapul-monary angiogenesis or is secondary to amelioration of cirrhosis It is worth noting that in vivo although we

found that 3-MA intervention alleviate the pulmonary pathological alterations, but the rats still presented obvi-ous ascites which is an indicator of Child-Pugh score We hypothesized that the effect of autophagy inhibition mainly comes from inhibition of intrapulmonary angiogenesis However, this hypothesis needs more data to be supported For example, we should use another administration method to demonstrate the targeted therapeutic effects, such as aerosol inhalation And, we should also explore the impact on liver

As we know, HPS is a really complex syndrome in which multiple factors change obviously Recently, it is hard

to answer which mechanism plays a dominant role in HPS In the early phases of CBDL, increased bilirubin, endotoxin and inflammatory mediators not only cause the liver injury but also are released into the circulatory system to damage distant organs These injuries further evoke the self-repair mechanisms, thus promoting the release of various growth factors and cytokines such as ET-1, TNF-α and VEGF-A Theses molecules activate the survival signals, such as Akt and ERK, which may in turn lead to ECs proliferation and pathological alterations of

Sham

2 weeks 4 weeks 2 weeks 4 weeks

Table 1 Effect of the autophagy inhibitor 3-methyladenine (3MA) on arterial oxygenation in 2- and 4-week CBDL rats * P < 0.05 compared with sham #P < 0.05 compared with CBDL 2 weeks ##P < 0.05 compared

with CBDL4 weeks Values are expressed as means ± SEM (n = 8) CBDL, common bile duct ligation; AaPO2, alveolar-arterial oxygen gradient

HPS24 Compared with these factors mentioned above, autophagy seems to be their common downstream which

is a cellular housekeeping process to remove damaged proteins and organelles through an alternative degradation mechanism (i.e., nonproteasomal) and serves as an adaptive response to maintain cell survival Further research

is needed to address mechanism difference attributed to autophagy and factors mentioned above

In conclusion, our study provides new insights into the role of autophagy in the pathogenesis of HPS From

our study of in vivo animal model, we demonstrated that an early intervention aimed at the reduction in

autopha-gosomes accumulation has the protective effects on HPS-associated pathological alterations and serious

hypox-emia From our study of in vitro cultured cell model, we further demonstrated that this protective effect might

result from the inhibition of PMVECs excessive proliferation We conclude that the initial overactive autophagy

Figure 4 Inhibition of autophagy with 3MA (5 mM) attenuated the excessive proliferation of PMVECs

induced by CBDL rat serum in vitro (A) Representative TEM images and graphical summaries of

autophagosomes in cultured PMVECs under different treatments in the absence or presence of 3MA Broad arrows represent autophgosomes Asterisks represent autolysosomes 30 random cells for each group were

observed (B) Western blotting and graphical summaries of LC3B, Beclin-1 and p62 protein levels in cultured

PMVECs under different treatments in the absence or presence of 3MA for 24 h All blots were representative

of three independent experiments (C) Representative immunocytochemistry images of LC3B, Beclin-1 and

p62 protein in cultured PMVECs under different treatments in the absence or presence of 3MA for 24 h

(D) Representative confocal microscope images and graphical summaries of LC3 in different groups of

PMVECs infected with mRFP-GFP-LC3 adenovirus for 24 h 30 random cells for each group were observed

(E) Cell viability in different groups of PMVECs was determined by CCK-8 analysis Data were from three

independent experiments Values were expressed as means ± SEM * P < 0.05.

may at least partly contribute to the development of HPS in CBDL rats (Fig 5) This finding may provide a new strategy for the clinical management of HPS or other proliferative vascular diseases

Materials and Methods Animal model Male Sprague-Dawley rats (200–220 g, 6 weeks, Third Military Medical University, Chongqing, China) were used in this study An experimental HPS rat model was successfully established by common bile duct ligation (CBDL) as previously described46,47 The experimental group underwent common bile duct ligation The control group underwent common bile duct exposure but no ligation The autophagy inhibitor 3-methyladenien (Sigma-Aldrich, St Louis, Missouri, USA) was administrated daily by intraperitoneal injection (15 mg/kg) within 2 weeks following CBDL The vehicle group animals were injected with the same volume of 0.9% saline Specimens were collected at the end of 2 and 4 weeks, respectively All rats were housed under a standard diet and living conditions (22–24 °C,12 h light/12 h dark cycle) All procedures performed on the animals were conducted according to the guidelines from the National Institutes of Health In addition, all experimental protocols were approved by the ethical committee of Third Military Medical University

Cell culture Cultured rat PMVECs were isolated from healthy Sprague-Dawley rats as previously described18,48 Cells were cultured in endothelial cell medium (ECM) with 10% fetal bovine serum (FBS), 100 U/ml

of penicillin-streptomycin and 1% endothelial cell growth supplement in a 95% O2/5% CO2 incubator at 37 °C PMVECs were incubated with 10% normal rat serum or 10% CBDL rat serum for 0 h (T1), 12 h (T2) and 24 h (T3) Experimental data were obtained from cells between passages third to six

Transmission electron microscopy Lung tissues and PMVECs were collected and fixed in 2% para-formaldehyde and 0.1% glutaraladehyde in 0.1 M sodium cacodylate for 2 h, post-fixed with 1% OSO4 for 1.5 h, washed, and stained for 1 h in 3% aqueous uranyl acetate And then lung tissues and cells were washed again, dehydrated by graded alcohol and embedded in Epon-Araldite resin (Canemco & Marivac, Quebec, Canada) Ultrathin sections were cut by an ultra-microtome (Reichert-June, Inc., Cambridge, UK), counterstained with 0.3% lead citrate and observed under a transmission electron microscope (model: EM420; Koninklijke Philips Electronics N.V., Amsterdam, The Netherlands)

Figure 5 Model of the initial autophagy activation and beneficial effects of 3-methyladenine (3-MA) intervention in rat experimental hepatopulmonary syndrome Pharmacological inhibition of autophagy with

3MA improved pathological alterations -pulmonary microvascular dilation and PMVECs excessive proliferation and typical symptom-hypoxemia in rat experimental hepatopulmonary syndrome.

Western blotting The protein samples extracted from lung tissues of rats or cultured PMVECs were sub-jected to SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA) Membranes were blocked for 1 h using 5% skim milk in TBST at room temperature, and then incubated with appropri-ate primary antibodies overnight at 4 °C (LC3B:1:1000, Beclin-1:1:1000, p62:1:1000; Abcam, Cambridge,

MA, USA), followed by incubation with secondary antibody (HRP-conjugated rabbit anti-goat IgG 1:10000; Abcam, Cambridge, MA, USA) Finally, the membranes were visualized using a gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA) The optical density of immnoreactivity was analyzed with an Alpha Imager (Protein Simple, San Francisco, CA, USA)

Immunofluorescence PMVECs were fixed with 4% formaldehyde for 30 min, permeability with 0.3% Triton X-100 for 10 min and blocked with 10% goat serum for 1 h at room temperature Cells were then incu-bated with appropriate primary antibodies overnight at 4 °C (LC3B:1:200, Beclin-1:1:200, p62:1:200; Abcam, Cambridge, MA, USA) followed by Alexa Fluor 488-labelled secondary antibody (Abcam, Cambridge, MA, USA) DAPI were used for nuclear staining (Beyotime Inc., Shanghai, China) Micrographs were obtained with a fluorescent microscope (Olympus BX51, Tokyo, Japan)

Autophagy detection using mRFP-GFP-LC3 adenovirus PMVECs were seeded onto cover slides and allowed to reach 50–70% confluence before transfection Adenoviral infection was performed according to the manufacturer’s instructions PMVECs were incubated in growth medium with mRFP-GFP-LC3 adenovirus (HanBio Technology Co., Shanghai, China) at 30 MOI After 12 h, the transfected cells were exposed to various indicated treatments Then cells were washed with PBS, fixed by 4% paraformaldehyde and analyzed by confocal microscope (Olympus, Tokyo, Japan)

Arterial blood gas analysis All animals were anaesthetized by intraperitoneal injection of sodium pento-barbitone (40 mg/kg) Arterial blood was drawn from the abdominal aorta and further analyzed using ABL 700 radiometer (Radiometer, Copenhagen, Denmark) The assessment of HPS in CBDL rats was based on the fol-lowing criteria: gas exchange dysfunction (PaO2 < 85 mmHg, A-aDO2 > 18 mmHg)49 Serum was separated from blood samples (7–8 ml), and then centrifuged at 2000× g/min in a Gyria for 10 min at 4 °C Following filtration with cellulose acetate membranes, serum was further inactivated at 56 °C for 30 min and stored at − 80 °C for use

in the subsequent experiments

Pulmonary wet-to-dry ratio Pulmonary edema was assessed by pulmonary wet-to-dry ratio as previ-ously described50 Briefly, lung tissues were weighed before and after storing at 80 °C for three days, and then the wet-to-dry ratio was calculated

Fluorescent-labeled microsphere assay Fluorescent-labeled microspheres (Life Technologies, Carlsbad,

CA, USA) in 0.2 ml of sterile distilled water were injected over 10 s through the jugular catheter, which was imme-diately flushed with 0.2 ml of sterile saline over 10 s After 30 min injection, lung and brain samples were collected and homogenized The fluorescent intensity was measured at 580/605 nm using a Multiscan Spectrum (Molecular Devices, Sunnyvale, CA, USA) And then, the ratio of brain/lung fluorescence intensity was calculated

Histological analysis Lung tissues were collected and fixed for histological analysis as previously described24 Briefly, after lung tissues were fixed in 10% formalin for 48 h, dehydrated in alcohol, embedded in paraffin, cut into 5-um thickness sections and stained with haematoxylin and eosin (H&E) The microphoto-graphs of the specimens were obtained with a light microscope (Olympus, Tokyo, Japan)

CCK-8 assay Cell proliferation was detected by the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan)

24 h after the same number of PMVECs was seeded in 96-well plates (0.8−1.0 × 104 cells per well), cells were pre-treated with autophagy inhibitor 3MA (5 mM) prior to the addition of DMEM containing different sera for the indicated time At the end of treatment, 10 ul CCK-8 solution was added to each well, and cells were cultured for

2 h at 37 °C After that, viable cells were detected by measuring the absorbance value at 450 nm using a Multiscan Spectrum (Molecular Devices, Sunnyvale, CA, USA)

Statistical analysis All data were expressed as the mean ± SEM and analyzed using SPSS 17.0 sta-tistical software (SPSS Inc., Chicago, IL, USA) Multiple comparisons between groups were analyzed with

Bonferron-i-corrected analysis of variance (ANOVA), and the remaining data were analyzed with Student’s t-test

A P value < 0.05 was considered statistically significant.

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